Viral replication is the process by which a virus (DNA or RNA) hijacks and uses the machinery of the cell it infects to multiply. By way of example, the main steps of the replication of retroviruses, and in particular of HIV viruses, are as follows: (1) fixing of the virus to the surface of a cell of an animal or human organism by recognition between virus surface proteins and receptors at the surface of said cell (for example the CD4 receptor); (2) penetration of the virus into the cell cytoplasm by fusion of the virus envelope with the cell membrane; (3) decapsidation of the virus (the virus separates from the matrix and from the capsid, which releases the two copies of the viral genome); (4) reverse transcription of the viral RNAs in the form of a proviral DNA by virtue of reverse transcriptase (viral enzyme); (5) migration of the proviral DNA into the nucleus and integration of that DNA into the DNA of the host cell under the effect of integrase (viral enzyme); (6) transcription of the DNA of the cell into genomic RNA (unspliced messenger RNA (mRNA)) under the effect of the RNA polymerase of the cell; (7) splicing of the mRNA, by excision of the introns, to leave only the exons (which code for the proteins Gag, Pol and Env); (8) translation, in the rough endoplasmic reticulum, of the mRNA in the form of polypeptides; (9) maturation of the polypeptides in the Golgi apparatus, allowing functional polypeptides to be obtained; (10) assembly of the viral particles at the surface of the membrane by accumulation of the multimerized structural polyproteins (Gag, p55), the viral nonstructural proteins (reverse transcriptase, integrase, protease) and the viral RNAs; (11) release of the virions by budding at the surface of the infected cell; and finally (12) maturation of the viruses.
Viruses have developed various strategies for escaping the immune system and facilitating their dissemination during the infection. In particular, the HIV virus has the particular feature of causing the complete breakdown of the immune system by attacking a key cell of the immune system, the auxiliary T lymphocyte (CD4+ T lymphocyte), which expresses at its surface the CD4 molecule, a specific HIV receptor. The monocytes-macrophages, the dendritic cells, the Langerhans cells and the cerebral microglial cells are likewise targets of HIV. The gradual disappearance of the lymphocytes leads to a lack of control of viral replication by the immune system, to the destruction of the lymphoid organs, where the immune response takes place, and to the onset of acquired immunodeficiency syndrome (AIDS), with the occurrence of severe opportunistic infections.
The mechanisms responsible for the disappearance of the CD4+ T lymphocytes during infection by HIV are complex, and they have been elucidated only partially.
The HIV viral particle is composed of a nucleocapsid which contains the single-stranded RNA dimer of positive polarity associated with the nucleocapsid protein, lysine tRNAs and the viral enzymes (reverse transcriptase, protease and integrase). The nucleocapsid is enclosed in a coat of matrix proteins which is covered by a lipid membrane borrowed from the host cell during budding of the viral particle. The membrane is provided with spikes composed of envelope glycoprotein oligomers. The step of conversion of the RNA into bicatenary DNA during the viral cycle under the action of a viral enzyme, reverse transcriptase, is the main characteristic of the retroviruses.
The viral genes gag, pol and env are retained in all retroviruses. All the products derived therefrom are present in the viral particle. They come from the cleavage of precursor polyproteins. The genes gag and env code for structural proteins, and the gene pol codes for numerous enzymatic proteins.
The Gag proteins are obtained from the cleavage of the polyprotein Pr55gag by viral protease. The cleavage releases the matrix protein, the capsid protein, the nucleocapsid protein, as well as a 6 kDa protein. Recent works suggest that these processes play an essential role in the genesis of infectious viral particles (Sticht et al., 2005; Ternois et al., 2005). These recent works have made it possible to generate, for the first time, an HIV assembly inhibitor.
The envelope precursor gp160 is cleaved into a surface glycoprotein gp120 (gp130 for SIVmac) and a transmembrane protein gp41 derived from the C-terminal region of the precursor. During its maturation, the precursor gp160 is glycosylated and then cleaved by a cell protease in the Golgi apparatus and then exported to the plasmic membrane. The two glycoproteins derived from the cleavage remain associated by non-covalent bonds. They form heteromers of envelope glycoproteins, which combine in oligomers to form the spikes of the virion.
The gene pol codes for three enzyme proteins: protease, reverse transcriptase and integrase. They are derived from the cleavage of the polyprotein Gag-Pol (Pr160 gag-pol) during the morphogenesis of the virion. Dimerization in the cell of the polyprotein Gag-Pol reveals the protease activity coded for by the 5′-region of pol. The mature form of the protease, released by autocatalytic cleavage, remains in the dimeric form p11/p11 and is then able to cleave other sites present on the polyproteins Pr160gag-pol and Pr55gag.
Reverse transcriptase is derived from the cleavage of the polyprotein Pr160gag-pol in two steps by the viral protease during the assembly of the viral particle.
Located in the C-terminal position of the Pol region of the polyprotein Gag-Pol, integrase is released in the form of a 32 kDa protein under the action of the viral protease. Its oligomerization is required both for its incorporation into the viral particle and to exert its activity of integrating linear double-stranded viral DNA into the cell genome.
All of these works emphasize the major role of protease(s) in the genesis of an infectious viral particle. Accordingly, as well as using retrotranscriptase inhibitors or nucleoside analogues, the HIV therapy known as highly active anti-retroviral therapy or “HAART” today includes one or more HIV protease inhibitors. This therapy leads to inhibition of viral replication, an increase in the number of CD4 T lymphocytes and an indisputable clinical improvement.
However, in so far as no current treatment enables patients to be cured of AIDS, and HIV virus isolates are or are becoming resistant to existing treatments, it is necessary to find other antiviral molecules which allow viral infections in general and infections by retroviruses such as HIV in particular to be combated more effectively.
Many viral infections coincide with disturbances in the mechanisms that control cell death (Barber, 2001). In particular, many works indicate that there is a relationship between in vitro or in vivo infection by HIV and an increase in the susceptibility to apoptosis of the T lymphocytes. Apoptosis (or programmed cell death or even cell suicide) is the process by which cells trigger their self-destruction in response to a signal (pro-apoptotic signal). The environment, interactions between cells, the absence of nourishment for the cell, infection by a pathogenic agent, are a few examples of pro-apoptotic signals. Apoptosis is a morphologically and biochemically defined form of cell death which is characterized in vivo by the absence of an inflammatory response, the activation of caspases and the cleavage of numerous proteins, fragmentation of the DNA, condensation of chromatin, cell contraction and the disassembly of cell structures to form vesicles incorporated into the membrane (apoptotic bodies). In vivo, this process culminates in the phagocytosis of apoptotic bodies by other cells.
Precocious apoptosis of a cell infected by a virus can constitute a defence mechanism of the host; it allows the number of viral particles released to be limited by interrupting viral replication. The cell endonucleases produced during apoptosis can act on the viral DNA and inhibit the synthesis of viral, structural and regulatory proteins and the formation of infectious viral particles, thus limiting the dissemination of virions in the host.
Accordingly, many viruses act on the regulation of the apoptotic intracellular signals, either in order to keep themselves alive or to keep the infected cell viable or to prevent the cell from being attacked by the effector cells of the immune system, and thus increase the efficacy of viral replication and permit greater production of virions. The majority of viruses have one or more genes permitting the synthesis of proteins whose effect is to suppress, at different stages, apoptosis of the cells they infect (antiapoptotic proteins). By retarding or inhibiting the death of the host cell, viruses promote the survival of the cell they infect and therefore their own survival, to the extent of promoting the occurrence of cancers in some cases.
Other viruses, on the other hand, have also developed strategies for causing the death of the cells they infect, leading to cell deficiencies, in particular immune deficiencies (such as those associated with AIDS), neuronal deficiencies (such as those associated with rabies) and epithelial deficiencies (such as those associated with haemorrhagic fevers). In the case of immune deficiencies alone, the viruses are then able to propagate. Some viruses are additionally capable of inducing apoptosis at a late phase of the infection, which allows the virions to propagate into the neighbouring cells while escaping the inflammatory and immune response of the host (Everett & McFadden, 1999).
The apoptotic processes induced by viruses are at the origin of cell disturbances which influence the clinical evolution of viral infections. Infection by HIV is a very representative example. Infection by HIV-1 is accompanied by abnormal induction of apoptosis in the adult T lymphocytes, the thymocytes and the haematopoietic precursors. Moreover, in a large number of patients, excess apoptosis affects all the lymphocyte populations (CD4 T, CD8 T and B), and the degree of apoptosis correlates with the evolution of the disease (Gougeon et al., 1996). In addition, in some patients infected by HIV, lymphocyte apoptosis is abnormally elevated in the lymphatic ganglions (Amendola et al., 1996), which constitute the main replication sites of the virus. Most surprising is that, in patients infected by HIV, the majority of the T cells that undergo apoptosis are not infected by the virus (“bystander” effect) (Finkel et al., 1995). These observations suggest that the destruction of the lymphocytes by HIV is the result of the activation by the virus of cytopathogenic mechanisms which are both direct and indirect.
The regulation of apoptosis by HIV is all the more complex because the virus is capable of manipulating the apoptotic machinery to its advantage by acting as both an activator and a repressor of apoptosis. HIV has in fact also developed mechanisms of inhibiting apoptosis in order to escape the host's immune system.
Various studies have shown that other lentiviruses, in particular feline immunodeficiency virus (FIV) and some strains of simian immunodeficiency virus (SIV), are also capable of inducing apoptosis and causing an immune deficiency syndrome in their natural host. Accordingly, within the scope of a study of different models of chronic lentiviral infections in primates, the inventors of the present invention have previously shown that the CD4+ T lymphocytes are abnormally sensitive to apoptosis in the rhesus macaque infected by the pathogenic strain SIVmac251 (Hurtrel et al., 2005).
On the other hand, in models of chronic lentiviral infections of primates in which the infection, whatever the lentiviral isolate in question, does not cause AIDS (chimpanzees experimentally infected by HIV, African green monkeys naturally infected by SIVagm), there is no abnormal programming of apoptosis of the CD4+ T lymphocytes in vitro. However, this absence of disease is not linked to the absence of pathogenic potential of the virus, since these viruses are capable of inducing AIDS in macaques (Hurtrel et al., 2005). Accordingly, these models of lentiviral infections underline the importance of factors proper to the host, which will determine either the occurrence of apoptosis associated with the development of AIDS, or the absence of pathology.
One of the major components of the machinery of apoptosis is a family of cysteine proteases called caspases (from the English cysteinyl aspartate-specific proteases or cysteine aspartate proteases). Caspases have been found in many organisms, ranging from C. elegans to humans. To date, more than about twelve caspases have been identified. These intracellular enzymes have a key role in apoptosis, inflammation, activation and cell differentiation.
Like other proteases, caspases are expressed in the form of proenzymes which undergo proteolytic maturation. These precursors are expressed constitutively in the cell cytoplasm. The procaspases (from 30 to 50 kD) contain three domains: an N-terminal prodomain, a large subunit (about 20 kD) and a small subunit (about 10 kD). Activation involves proteolytic cleavage between the domains, followed by the association of the large and the small subunit, each of which contributes to the amino acids of the active site, to form a heterodimer. The active mature enzymes function in the form of a tetramer composed of two heterodimers. The N-terminal domain of the caspases, whose length (from 23 to 216 amino acids) and sequence vary greatly, is involved in the regulation of those enzymes.
The function of the caspases is determined by their substrate specificity, the length of their prodomain and the sequence of the prodomain. The caspases can be divided into three groups: the inflammatory caspases (group I), the initiator (or regulatory) caspases (group II) and the effector (or executor) caspases (group III) (Lavrik et al., 2005). The long prodomain (more than 100 amino acids) of the initiator caspases and of the inflammatory caspases acts as an apoptotic or proinflammatory signal integrator. The inflammatory caspases include caspase-1, -4, -5, -11, -12, -13 and -14. They are involved in the inflammatory processes and play a central role in the activation of certain cytokines. The initiator caspases include caspase-2, -8, -9 and -10. They are located upstream of the apoptotic signalling cascades and are activated by autoproteolytic mechanisms in response to proapoptotic signals. They then cleave and activate the effector caspases, which are located downstream of the signalling cascades, permitting amplification of the apoptotic signal. The effector caspases include caspase-3, -6 and -7. They are involved directly in the execution or occurrence of apoptosis; once activated by the initiator caspases, they cleave numerous cell proteins, thus leading to dismantling of the cell or inactivation of other proteins (Thornberry and Labzebnik, 1998). The proteins inactivated by the action of these caspases (approximately from 2000 to 3000 substrates) include proteins which protect the cells from apoptosis (antiapoptotic proteins), such as proteins of the Bcl-2 family.
The caspases, the catalytic domain of which includes a cysteine residue (C), cleave their protein substrate(s) at specific consensus sites containing an aspartic residue which are located in the carboxy-terminal part of the substrate. They exhibit substrate recognition motifs and highly conserved catalytic motifs (Cryns et al., 1998).
The preferences or substrate specificities of individual caspases have been used to develop peptides which effectively enter into competition with the binding of the caspases to their substrate. Such synthetic inhibitors are now available commercially. Some broad-spectrum caspase inhibitors include a single amino acid or a generally di- to tetra-peptide amino acid sequence, which is optionally O-methylated and which is conjugated to a carboxy-terminal group such as fluoromethyl ketone (fmk), chloromethyl ketone (cmk), an aldehyde group (CHO) or a difluorophenoxy group (OPh). Such inhibitors have been described especially in patent application WO 02/183341. These caspase inhibitors are capable of penetrating the cells and binding irreversibly (with the exception of inhibitors having an aldehyde group, whose binding is reversible) to the active site of the caspases. They accordingly act as proteolytic decoys by blocking proteolytic caspase cleavage, which is required for activation of said caspases and the production of an active truncated caspase. Inhibitors having a carboxy-terminal group fmk or OPh have been formulated for in vivo and in vitro applications.
Two of the most widely used caspase peptide inhibitors are the inhibitors Boc-D-fmk (tert-butyloxycarbonyl-Asp(O-methyl)-fluoromethyl ketone); Enzyme Systems Products, CalBiochem or R&D Systems) and z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; Enzyme Systems Products, CalBiochem or R&D Systems). The Boc (tert-butyloxycarbonyl) and z (N-benzyloxycarbonyl) groups serve to block the amino acid sequences D (Asp) or VAD (Val-Ala-Asp), while the fluoromethyl ketone group in the carboxy-terminal position facilitates membrane permeability. It has been shown that inhibition of the activation of caspases by such inhibitors prevents the appearance of the morphological modifications which are characteristic of apoptosis (Chinnaiyan et al., 1997).
A more recently developed caspase inhibitor, Q-VD-OPh (N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxyl)methyl ketone); Enzyme Systems Products, CalBiochem or R&D Systems), has increased efficacy, stability and permeability as compared with inhibitors having a carboxy-terminal group of the fluoromethyl ketone (fmk) type, and reduced toxicity, even when used in a high concentration; this inhibitor has been found to be non-toxic at doses of up to 1 g/kg live weight, when administered to mice by the intraperitoneal route (Vera et al., 2005).
The inhibitor Q-VD-OPh has been found to be functional in vitro, in different cell types, and in vivo, in animal models, in particular in the mouse and the rat. Moreover, it has been shown that it inhibits various caspases, in particular caspase-1, -3, -8, -9, -10 and -12, with IC50 values ranging from 25 to 400 nM. Furthermore, Q-VD-OPh, like the inhibitors ZVAD-fmk and Boc-D-fmk, inhibits apoptosis in a dose-dependent manner (Caserta et al., 2003).
It has been proposed that the inhibition of caspases or the overexpression of the antiapoptotic protein Bcl-2 might prevent viral infections by inhibiting apoptosis, and also disrupt viral production (Levine, 1996; Olsen, 1996; Liang, 1998; Wurzer, 2003). However, studies conducted in vitro using the caspase inhibitor z-VAD-fmk did not enable viral replication to be inhibited (Gandhi et al., 1998; Petit et al., 2002). On the contrary, administration of the caspase inhibitor z-VAD-fmk to T-leukaemia cells (CEM) or to peripheral blood mononuclear cells (PBMC) exposed to the HIV-1 virus had the effect of increasing viral replication (Chinnaiyan et al., 1997). Similar results have been observed in the case of CEM cells expressing the CrmA (cytokine response modifier A) protein of the vaccinia virus, a viral caspase inhibitor that inhibits especially activation of caspase-1, -6 and -8 (Chinnaiyan et al., 1997). This suggests that the results observed in the case of the inhibitor z-VAD-fmk are not specific to that inhibitor but rather the result of the inhibition of proapoptotic proteases. Furthermore, the inhibitor z-VAD-fmk is capable of stimulating endogenous viral replication in activated PBMCs derived from patients who are HIV-1-positive but are asymptomatic (Chinnaiyan et al., 1997). The totality of these results suggests that apoptosis might help the host to limit propagation of the virus and that, consequently, strategies aimed at inhibiting cell death might have deleterious consequences for the infected host and might, in particular, contribute towards increasing the viral load of an HIV-positive individual. This would be in agreement with the fact that many viruses produce proteins that inhibit cell death in the host (for example the CrmA protein produced by the vaccinia virus).